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The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsieila oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, d-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, [CSFp.sup.OmlA]-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. [CSFP.sup.OmlA]-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in [CSFP.sup.OmlA]-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed [Asp.sup.+2] and [Lys.sup.+3] [Ser.sup.+4] function as inner membrane retention signals and identified four novel inner membrane retention signals: [CK.sup.+2] [V.sup.+3] [E.sup.+4], [CG.sup.+2] [G.sup.+3] [G.sup.+4], [CG.sup.+2] [D.sup.+3] [D.sup.+4], and [CQ.sup.+2] [G.sup.+3] [S.sup.+4]. These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing [PA4370.sup.CGDD-ChFP] and [PA3262.sup.CDSQ]-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The [beta]-galaetosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all see genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the See-dependent export of periplasmic Male and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane See translocase by SRP.

Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini.

The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of [beta]-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pill and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pill. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic 'pseudopilus' and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).

Results show that the Klebsiella oxytoca cells defective in secretion of pullulanase, because of a mutation in the gene encoding a periplasmic disulfide oxidoreductase pullulanase, are rectified by the overproduction of pseudopilin PulK. Data indicate that pseudopilin contains reduced disulfide bonds, which impair the secretion process.

Results demonstrate that in the dynamic interactions between components for the pullulanase secretion, PulL specifically stabilizes and anchors PulE to the cytoplasmic membrane. Data indicate that not all components of the protein are required for secretory protein recognition and its specificity.

Research has been conducted on the Escherichia coli chromosomal genes. Results indicate that E. coli piliation genes have been expresed at low levels.

A study concerning a possible domain structure of the dodecameric outer membrane secretin from Klebsiella oxytoca is analyzed. Results indicate that beta domain is a determinant of multimer stability.

Expression of the Escherichia coli K-12 operon gene gspO using the lacZ promoter on plasmids has identified a protein similar to several type IV prepilin peptidases. The gene induced the cleavage of two known prepilin peptidase substrates. Meanwhile, expression of the pulG homolog gspG has resulted in the production of a specific protein recognized by the PulG antiserum.

Pullulanase secretion from Escherichia coli containing secretion genes from Klebsiella oxytoca was not inhibited by 23 separate linker insertions of 2-4 codons each, but was inhibited by fusion of reporter proteins to either the N- or C-terminus. Shortening one reporter protein, however, did permit secretion. Eleven of the insertions abolished pullulanase activity. The reporter hybrids did not alter activity, suggesting that pullulanase conformation was not significantly changed in these hybrids.

The TnphoA mutagenesis, to create a number of irp2-phoA gene fusions on a multicopy number plasmid, and then by determining the conditions under which HMWP2 does not form disulfide-bonded aggregates when the cells were disrupted are the processes which proved that HMWP2 is a predominantly soluble cytoplasmic protein with a cryptic internal export signal in Yersinia enterocolitica. This high alkaline phosphatase activity is associated with some of the HMWP2-PhoA hybrids.

PCR amplification method yields the N. gonorrhoeae prepilin gene, pilD-Ng, which splits and subsequently N-methylates the precursor pilin subunits before assembling into pili. Accurate splitting of the gonococcal prepilin in Escherichia coli cells containing the prepilin peptidase gene and the pilin structural gene is promoted by the pilD-Ng product.

The high-molecular-weight protein 2 (HMWP2) of Yersinia enterocolitica is identical to AngR of Vibrio anguillarum in its structure, and may be involved in the nonribosomal preparation of small biologically active peptides. The nucleotide sequencing of the iron regulated irp 2 gene and its promoter region reveals the role of HMWP2, which is identical in many respects to the superfamily of enzymes that form adenylate. The irp2 gene, found exclusively in disease causing Yersinia species, encodes HMWP2.

Protein transport across the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) requires hydrophobic amino acids. The translocation of hydrophilic polypeptides needs at least six different sec gene-encoded proteins and is dependent on ATP hydrolysis and the electrochemical potential. Other variants of the GSP terminal branch have been identified and presented.

The gene (cpo) encoding the extracellular protease CPI produced by the moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 is cloned and its nucleotide sequence is studied. The cpo gene has encoded a 733-residue protein and the type II secretion apparatus is responsible for the secretion of the haloprotease CPI.

Proteins called secretins form large multimeric complexes that are essential for macro molecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic. [ABSTRACT FROM AUTHOR]